The long term objective of this project is to understand the role that lipids (including retinal) play in the function of biological membranes. Specific questions addressed are: What is the role of the highly unsaturated fatty acids (PUFA) found in the phospholipids of rod outer segment (ROS) membranes? What are the effects of oxidized lipids on membrane structure? What are the molecular interactions of retinal with amino acid side chains in rhodopsin and bacteriorhodopsin before and after light excitation? There is physiological evidence that highly unsaturated lipids play an important role in ROS and other membranes; however, the mechanism is unknown. 2H-NMR will be used to study protein-lipid interactions by reconstituting rhodopsin with specifically deuterated lipids. A new concept for the biological function of 22:6 fatty acids, coupling functional membrane protein conformational changes with lipid area and thickness changes, will be tested using 2H-NMR and FTIR. The role of different lipid head groups (PC, PE, PS and PA) and ROS G protein on modulating 22:6 action will be investigated using 2H-NMR, 14N-NMR and 13P-NMR as well as FTIR. Highly unsaturated lipids are very easily damaged by oxidation, and the products are toxic to cells. The effects of oxidized lipids on membrane structure and dynamics will be studied using NMR methods. Solid state NMR methods will be used to study 13C labeled retinals will be incorporated into rhodopsin and bacteriorhodopsin in native membranes in collaboration with the Nakanishi research group at Columbia. Retinals specifically labeled with 19F will be incorporated into rhodopsin and bacteriorhodopsin in collaboration with Robert Liu at the University of Hawaii and studied by solid state NMR. Solid state NMR methods will be used to map out the detailed interactions between retinal and amino acid side chains of rhodopsin and bacteriorhodopsin after light excitation by low temperature trapping of intermediates. The state of protonation of aspartic acids will be studied by solid state NMR at key points after light excitation by incorporating carboxyl 13C-labeled Asp into bacteriorhodopsin.